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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 8 (1987), S. 312-323 
    ISSN: 0886-1544
    Keywords: plant cytoskeleton ; Chlamydomonas ; anti-IFA ; onion root tip cells ; immunoflurescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Four monoclonal antibodies were raised against polypetides present in a highsalt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunoflurescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antiboides labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with varibale intensites. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,0000 Mr (two to three bands) polypetides and a diffuse and around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypetides putative plant intermediate filament proteins.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 24 (1989), S. 205-218 
    ISSN: 0148-7280
    Keywords: oocyte aging ; immature oocytes ; oocyte maturation ; chromosome abnormalities ; in vitro fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: It has been suggested that the abnormal maturation of the human oocyte during fertilization in vitro may result in chromosome imbalance and, induce embryonic loss. Using a mouse model, we have studied the influence of the degree of oocyte maturation (either immaturity or overmaturity) on the chromosome characteristics of embryos at the first-cleavage division. Immature oocytes were obtained 2-3 h or 3-4 h before the expected ovulation time (b.o.). Overmaturation was induced by aging the newly ovulated oocytes in vitro for 3,6, and 12 h. Our results show a significant decrease in the fertilization rate in the immature groups (65.53% at 2-3 h b.o. and 16.59% at 3-4 h b.o. vs. 78.22% at control) and after 12 h of in vitro aging (69.39%), while a significant increase of this parameter was found at 3 h of aging (82.59%) as compared to the other groups. No significant differences were found in the occurrence of aneup'.oidy or hypcrhaploidy in embryos obtained from immature, newly ovulated, and overmature oocytes. Finally, an increased incidence of polyploidy was detected in immature, 2-3 h b.o. (31.20%), and overmature, 3 h (23.04%) and 6 h (31.61%), groups as compared to the control group (14.59%).
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 93-111 
    ISSN: 0148-7280
    Keywords: sperm-oocyte fusion ; reactive oxygen species ; motility ; calcium ; cAMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The possible role of calmodulin in regulating a number of calcium-dependent functions exhibited by human spermatozoa was investigated by using the antagonists trifluoperazine and calmidazolium. At high doses both antagonists inhibited the motility of human spermatozoa and induced a concomitant rise in [Ca2+]i and a decline in cAMP. Lower doses of these antagonists, particularly calmidazolium, suppressed the ability of human spermatozoa to generate reactive oxygen species and exhibit sperm-oocyte fusion, without influencing [Ca2+]i, cAMP, or motility. This inhibition of sperm-oocyte fusion was effective even if the spermatozoa were subsequently exposed to A23187, suggesting that calmodulin may regulate this aspect of human sperm function at a point downstream from calcium influx.Both radiolabelling and affinity chromatography techniques were used to detect a number of calcium-dependent and calcium-independent calmodulin acceptor proteins in the human spermatozoon. The major calcium-dependent acceptor proteins exhibited Mr values of 32,000 and 22,000-27,000, respectively, and did not appear to be associated with the sperm plasma membrane.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 185-192 
    ISSN: 0148-7280
    Keywords: oocyte ; fusion ; protein synthesis inhibition ; maturation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The method of polyethylene-glycol-induced fusion of mammalian oocytes was applied to study maturation-promoting factor (MPF) activity. After homologous fusions of one maturing-late diakinesis (LD), metaphase I (MI)-pig or mouse oocyte to one, two, or three immature-germinal vesicle (GV)-oocytes, giant cells were cultured in control or cycloheximide supplemented medium for 3 hours. The occurrence of germinal vesicle breakdown (GVBD) and premature chromosome condensation (PCC) served as a control of MPF activity. In giant cells composed of one maturing and one, two, or three immature oocytes, GVBD and PCC were observed in all cases after cultivation in the control medium. In the presence of cycloheximide, the completion of GVBD and PCC remained high when one maturing and one immature oocyte were fused (83.7% and 95.7% of GVBD in pig and mouse, respectively). However, in giant cells composed of one maturing and up to three immature oocytes, all GVs were broken down only occasionally (4.8% and 11.7% in pig and mouse, respectively). These results suggest that in pig and mouse oocytes MPF does not amplify autocatalytically, but requires active protein synthesis for its production.
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  • 5
    ISSN: 0886-1544
    Keywords: nuclear migration ; microtubules ; F-actin ; root hairs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A prominent feature of tip growth in filamentous plant cells is that the nucleus often migrates in step with the tip as it extends. We have studied this long-recognized but unexplained relationship in root hairs of the legume Vicia hirsuta by a variety of microscopic techniques. Using rhodaminyl lysine phallotoxin, and antitubulin antibodies, root hairs are shown to contain axial bundles of F-actin and a complex microtubular system. To the basal side of the nucleus the microtubules are cortical and net axial but in the region between nucleus and tip the arrangement is more complicated. Electron microscopic thin sections demonstrate that internal bundles of microtubles exist in addition to the plasma membrane-associated kind. Computerized deblurring of through-focal series of antitubulin stained hairs clarifies the three-dimensional organization: bundles of endoplasmic microtubules progress from the nuclear region toward the apical dome where they can be seen to fountain out upon the cortex.The relationship between nucleus and tip can be uncoupled with antimicrotubule herbicides. Time lapse video microscopy shows that these agents cause the nucleus to migrate toward the base. This contrary migration can be inhibited by adding cytochalasin D, which fragments the F-actin bundles.It is concluded that microtubules connect the nucleus to the tip but that F-actin is involved in basipetal migration as is known to occur when symbiotic bacteria uncouple the nucleus from the tip.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 8 (1987), S. 360-367 
    ISSN: 0886-1544
    Keywords: spindle ; autoantibody ; CREST ; scleroderma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: An autoantibody that binds an antigen localized to the stembody of dividing cells has been identified in a patient with systemic sclerosis. Initially, this antigen is associated with the surface of the metaphase chromosomes. At the onset of anaphase the antigen becomes preferentially associated with the forming stembodies. This association is maintained as furrowing progresses during telophase and continues after the intercellular bridge is released from the daughter cells during G-1. Immunoblots indicate that the epitope detected by immunoflurorescence is present on a protein with an apparent molecular weight of 38 kD.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 18 (1987), S. 97-107 
    ISSN: 0148-7280
    Keywords: protein synthesis ; oocyte ; human ; electrophoresis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: As a first step toward understanding control of gene expression in early human development, an analysis of protein synthesis and amino-acid transport in unfertilized mature oocytes was initiated. Qualitative patterns of protein synthesis were examined in individual oocytes cultured in medium containing radiolabeled methionine. No differences in synthetic pattern of proteins, resolved by one-dimensional electrophoresis and fluorography, were observed in oocytes analyzed from times varying from 12 to 52 hr following collection by laparoscopy. Contamination by follicular or corona radiata cells was readily distinguished on the basis of increased relative synthesis of a polypeptide with Mr = 44,000, a dominant product of synthesis in follicular cells. Based on the specific activity of the methionine precursor, the absolute rate of synthesis was measured to be about 50 pg/oocytc/hr, a value higher than in mouse unfertilized eggs. No difference in protein synthetic rate was observed in oocytes analyzed at 12 hr postcollection versus later times up to 50 hr postcollection. Competition of methionine uptake by leucine, efflux of radiolabeled methionine from preloaded oocytes into medium containing methionine and uptake of methionine in medium with low sodium ion concentration was observed. These findings are consistent with the presence of an L (leucine-preferring) system for neutral amino acid transport, similar to that in mouse and rabbit eggs. Total protein was measured to be about 150 ng/oocyte, a value five times that of the mouse. These studies provide basic data for further analysis of oocytes and perhaps preimplantation stage embryos in the future.
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  • 8
    ISSN: 0148-7280
    Keywords: heparin ; fertilization ; dextran sulfate ; fucose sulfate glycoconjugate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P 〈0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.
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  • 9
    ISSN: 0192-253X
    Keywords: ecdysteroid ; prothoracic gland ; temperature sensitive ; Drosophila ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The dominant temperature-sensitive mutation L(3)3DTS (DTS-3) in Drosophila melanogaster causes lethality of heterozygotes during the third larval instar at the restrictive temperature (29°C). Temperature-shift experiments revealed two distinct temperature-sensitive periods, with lethal phases during the third larval instar (which may persist for 4 weeks) and during the late pupal stage. At 29°C mutant imaginal discs are unable to evert in situ, but did evert normally if cultured in the presence of exogenous ecdysterone or when implanted into wild-type larval hosts. The only morphologically abnormal tissue present in the lethal larvae is the ring gland, the prothoracic gland being greatly hypertrophied in third instar DTS-3 larvae. Injection of a single wild-type ring gland rescued these mutant larvae, indicating that the mutant gland is functionally, as well as morphologically, abnormal. Finally, the mutant larvae were shown to have less than 10% of the wild-type ecdysteroid levels. These results are all consistent with a proposed lesion in ecdysteroid hormone production in DTS-3 larvae. A comparison with the phenotypes of other “ecdysone-less” mutants is presented.
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  • 10
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Molecular cloning of the genes encoding the muscarinic acetylcholine receptors has shwon that receptor subtypes classified on the basis of pharmacological properties are related polypeptides encoded by distinct genes. These studies have laso revealed the existence of novel muscarinic receptor subtypes. Functional analysis of each of the subtypes expressed in mammalian cells indicates that the different subtypes activate distinct biochemical pathways, a finding that explains the tissue-specific physiological response elicited by the neurotransmitter, acetylcholine.
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